Microbial manipulation of host SNARE machinery is an emerging field in cellular microbiology. My research focuses on the mechanisms that the obligate intracellular bacterium Chlamydia trachomatis employs to hijack sphingolipid metabolites from the host cell. C. trachomatis is an obligate intracellular pathogen that replicates within a parasitophorous vacuole termed an inclusion. Sphingolipids are required for the integrity of the inclusion membrane (IM) and are also incorporated into the chlamydial cell wall, indicating their importance in both chlamydial development and viability. The mechanisms by which the IM and, thus, chlamydiae obtain eukaryotic lipids are poorly understood. Previous studies have shown that the inclusion intercepts Golgi-derived exocytic vesicles containing sphingomyelin (SM) and cholesterol. To examine Golgi-derived vectoral trafficking to the chlamydial inclusion, a polarized epithelial cell model was developed. Consistent with previous studies, SM was retained within chlamydial-infected cells and associated with purified C. trachomatis elementary bodies. The retention of SM by chlamydiae correlates with a disruption of basolateral SM trafficking, suggesting that chlamydiae preferentially intercept basolaterally-directed, SM-containing exocytic vesicles. This proposal is designed to elucidate mechanisms of chlamydial lipid acquisition by specifically investigating the role of a trans-Golgi associated soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) protein syntaxin 6 in this process. Preliminary data demonstrate that syntaxin 6 localizes to the chlamydial inclusion. This process requires chlamydial protein synthesis and is conserved across species, similar to chlamydial recruitment and retention of SM. Our central hypothesis is that syntaxin 6 mediates chlamydial lipid acquisition. In Specific Aim 1, we will determine the role of syntaxin 6 in chlamydial SM acquisition. To this end, we will examine SM trafficking to the inclusion with established intracellular lipid trafficking protocols in syntaxin 6 knock down cells, identify additional lipids that syntaxin 6 may be trafficking to the inclusion, and identify chlamydial binding partners for syntaxin 6. The localization of syntaxin 6 to the chlamydial inclusion requires a tyrosine motif or plasma membrane retrieval signal (YGRL). In Specific Aim 2, we will characterize the role of the syntaxin 6 YGRL motif in the targeting of proteins to the chlamydial inclusion. To this end, we will examine if other eukaryotic proteins containing the YGRL motif localize to the chlamydial inclusion and if addition of the YGRL causes retargeting of other eukaryotic proteins to the inclusion. Importantly, these studies will define a mechanism for the elusive, but critical process of chlamydial lipid acquisition. Additionally, these studies may identify a eukaryotic signal sequence that targets eukaryotic proteins to an intracellular bacterial parasitophorous vacuole. Public Health Relevance: The obligate intracellular pathogen Chlamydia trachomatis, a serious human pathogen, requires host-derived lipids, such as sphingomyelin, for survival. These studies will examine the role of syntaxin 6 in the poorly understood process of chlamydial lipid acquisition and define a mechanism for lipid acquisition.